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RNA quality is a critical determinant for the success of many downstream applications, including microarray analysis. For this reason, if you are planning microarray analysis experiments, it is important to fully consider the factors that affect the quality of your RNA.
Tissue-Specific Responses to Injury
Affect RNA Integrity
Traditionally, efforts to preserve RNA quality
have focused on methods of tissue storage and disruption, with
the goal of minimizing RNase activity. However, a more
critical determinant is actually the RNA quality within
tissues before the RNA expression pattern is 'frozen' by
preservation. RNA integrity within the cell is dependent on a
complex series of responses that are set in motion in response
to insult, as well as the interval between the time of injury
and tissue preservation. The mechanisms of RNA degradation can
be both complex and varied, as is the resulting impact on the
mRNA population. Therefore, no matter how carefully you
prepare your RNA, the integrity is often determined before the
tissue sample reaches your hands.
Residual Contaminants -- The Hidden
RNA Quality Factor
Even the
most intact RNA will not perform well if the sample contains
trace contaminants. The most detrimental contaminants are
residual organics, metals, and proteins such as nucleases. RNA
that contains these impurities will perform poorly in most
enzymatic applications.
Residual contaminants are most often a problem in RNA isolated with single-step organic extraction protocols. Although relatively fast and easy, single-step extraction may not be sufficient to remove contaminants from some tissues, especially if you exceed the recommendation for input sample amount. Organic contaminants are often carried over into samples during aqueous phase transfer; to avoid this, leave some of the aqueous phase behind during phase separation.
We recommend a combination of phenol based and solid phase extraction methods to avoid this inadvertent carryover of contaminants. Several studies have shown that RNA isolated by a combination of both techniques provides superior array data than RNA isolated by either method alone (1-3). Figure 1 compares the Agilent 2100 bioanalyzer profiles of RNA isolated with a single-step organic extraction method to RNA further purified using solid phase extraction with Ambion's MEGAclear Kit. In overnight stability tests, the 28S:18S rRNA ratio of the RNA isolated by the single-step protocol decreased 32% when incubated at 37C compared to the control stored at 20C (compare Figure 1, Panels A and B), indicating the presence of residual contaminants in the RNA. However, no change in the rRNA ratio was observed between the MEGAclear-purified samples stored at 20C and 37C (compare Panels C and D). These data illustrate the negative impact of residual contaminants on RNA integrity and their potential to inhibit downstream enzymatic applications. Therefore, if your RNA wa s obtained using a one-step method and has not performed well, we recommend the use of MEGAclear as a fast and convenient solid phase clean-up method.
Figure 1. A Combination of Single Step Organic Extraction and Solid Phase RNA Extraction Improves RNA Quality. RNA was extracted either using single phase organic extraction (A, B) or single phase organic extraction followed by purification with Ambion's MEGAclear Kit (C, D). The RNA was stored overnight at either 20C (A, C) or 37C (B, D).
Removing Genomic DNA and Small RNAs
Other contaminants that can affect total RNA performance
in downstream applications are residual DNA contamination and small RNAs
such as 5S and tRNAs. Residual genomic DNA contamination is most problematic
in tissues with high cell densities, such as spleen or tissue culture
cells. Figure 2 shows examples of gross contamination with genomic DNA.
High molecular weight genomic DNA typically migrates as a broad, larger
molecular weight peak that is well separated from rRNA peaks (Panel A).
Note, also, that the base-line is high in this electropherogram: this
is generally a signature of underlying genomic DNA contamination. Genomic
DNA that has been partially sheared can sometimes migrate between the
18S and 28S rRNA ribosomal bands (Panel B), making it difficult to accurately
determine the rRNA ratio. Incomplete DNase I digestion can generate small
molecular weight DNA fragments between 50200 bases in size (Panel
C). The most common causes of incomplete DNA digestion are residual contaminants
(high salt, residual organics, etc) that inhibit enzyme activity, or the
use o
f an insufficient amount of DNase I. To efficiently remove genomic
DNA, we recommend treating your samples with Ambion's TURBO DNase,
a DNase I with improved activity. MEGAclear can also be used to rapidly
purify RNA following DNase I treatment.
Figure 2. Different Types of Genomic DNA Contamination In Total RNA Preparations. Agilent 2100 bioanalyzer electropherograms of RNA contaminated with high molecular weight genomic DNA (A), partially sheared genomic DNA (B), and small DNA fragments generated by incomplete DNase I digestion (C).
Small RNAs, such as 5S and tRNAs, are
efficiently recovered in organic extraction methods but are
depleted in column-based purification methods (compare Panels
A and C, and Panels B and D in Figure 1). At Ambion, we have
found that total RNA samples in which small RNAs have been
removed by solid phase extraction (following organic
extraction) show superior performance in RNA amplification
compared to RNA isolated by organic extraction only. Since
small RNAs can comprise as much as 15% of total RNA, their
removal effectively increases the percent of mRNA within the
total RNA sample and decreases the potential for interference
during cDNA synthesis.
The rRNA Ratio Should Not Be Used as the
Sole Indicator of mRNA Quality
The 28S:18S rRNA ratio has traditionally been viewed as
the primary indicator of RNA quality, with a ratio of 2.0
considered to be indicative of high quality, intact RNA.
However, with widespread use of the Agilent 2100 bioanalyzer,
it has become increasingly clear that the long time standard
of a 2.0 rRNA ratio is difficult to meet, especially in RNA
derived from clinical samples. This has led researchers to
question the wisdom of using the ratio of the 28S and 18S
rRNAs, two highly structured and long-lived molecules, as the
sole measure of the quality of the underlying mRNA. At Ambion,
we find that total RNAs with 28S:18S rRNA ratios of 1.0 or
greater usually provide high quality intact mRNA, and perform
well in a variety of applications.
One way to extrapolate information about sample integrity is to carefully evaluate the baseline in the bioanalyzer electropherogram. In high quality RNA (rRNA ratio near 2.0), the baseline above and below the 18S and 28S rRNA peaks will be relatively flat. As the 28S rRNA breaks down, the degradation products will cause the baseline between and below the 18S and 28S rRNA peaks to rise. The 18S and 28S rRNAs will appear to be riding on top of the baseline.
As a cautionary note, some total RNAs will often contain classes of small RNAs that may initially appear to be breakdown products but are actually abundant tissue-specific mRNAs (Figure 3). These abundant small RNAs are most often found in RNA isolated from reproductive and intestinal tissues. Their fluorescence can sometimes dwarf that of the rRNAs. Generally, they can be distinguished from degradation prod ucts because the rRNA ratio of the sample will be greater than 1.0 and the baseline between the small RNAs and the 18S RNA will be relatively low.
Figure 3. Low Baseline Fluorescence Indicates Good RNA Quality.(A) Partially degraded total RNA. The 28:18S rRNA ratio is 0.9 and the baseline fluorescence is elevated both between the 28S and 18S and below the 18S, making the rRNA peaks appear to be riding on top of the baseline. (B) Human intestinal RNA containing abundant small RNAs. This sample can be distinguished from partially degraded RNA by the relatively high 28S:18S rRNA ratio, and the relatively low baseline both between the 28S and 18S peaks, and immediately below the 18S peak.
Our data suggest that, aside from
integrity, trace contaminants may be the largest contributor
to poor performance in sensitive enzymatic applications such
as amplification for microarray analysis. Most often
impurities interfere with cDNA synthesis steps, resulting in
reduced size and yield of aRNA following in vitro
transcription. In fact, a recent study on the impact of
moderate RNA degradation on microarray analysis suggests that
while the loss of the 5' end of transcripts results in higher
ratios of hybridization to 3' end probes than 5' end probes,
the number of genes detected or differentially expressed is
not significantly reduced (4). Stated simply, RNA quality can
be defined as the sum of RNA integrity and RNA purity. Many
researchers are finding that their application may be tolerant
of some loss in
RNA integrity, as long as their RNA is free of
residual contaminants.
Ordering Information
Cat#
Product Name
Size
1907
TURBO DNA-free
50 rxns
1908
MEGAclear Kit
20 rxns
2238
TURBO DNase (2 U/l)
1000 U
2239
TURBO DNase (2 U/l)
5000 U
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Source:
Related biology technology :
1. A Unique Dimension in Biological Purity
2. Oligonucleotide Purity Analysis by Capillary Electrophoresis
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